The publications and other materials used herein to illustrate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Trends in Diagnostic Testing
Wide variety of methods and instruments are commercially available for in vitro immunodiagnostic (IVD) testing of clinical samples. Traditional IVD tests, such as ELISA immunoassay tests, are characterized with complicated test methodology. A test may need addition of reagents in several steps and washing in several steps. This makes the tests laborious to perform. In order to reduce the need of labour, automated analysers have been developed. The analysers can work either in “random-access mode” or in “batch mode”. The automated analysers can run up to several hundreds of tests an hour. Typically, the larger the analyser, the higher the test capacity is. The test menu of an automated random-access analyser can contain tests up to 50 different analytes, or even more. By the economy of size, a large analyser can provide results cheaper than a small analyser. This has pushed IVD testing towards large centralized laboratories.
The main drawback of centralized testing is the long turn-around-time, which is far too long to satisfy the testing need of acute patient cases. Therefore, the trend of centralization has been followed by the trend of near-patient-testing, i.e. point-of-care testing. At the point-of-care, there is an increasing need for test instruments which provide rapid results. To be applicable in the point-of-care, the instrument should be easy to use, small in size, and affordable in price.
In order to meet with the requirements of point-of-care testing, the test methodology should be as simple as possible. A widely used approach for simplifying the test methodology is to apply dried (or lyophilised) biochemical reagents in place of liquid reagents. The use of dried reagents can eliminate the steps of reagent addition.
Another approach to simplify test methodology is to apply a detection technology which allows separation-free (wash-free) detection of bioaffinity assays. The use of a separation-free detection technique can eliminate washing steps.
An approach to reduce the size of the analyser is to reduce reaction volumes, i.e. to miniaturize the testing system. This also reduces volumes of test consumables, such as test reagents and buffers. This makes the test better suited for point-of-care use. Miniaturizing, however, usually compromises the performance figures of the detection technique. To avoid this, a detection technique which tolerates miniaturization without compromising performance should be used.
Dried Reagents
It is widely known that bioaffinity reagents, such as antibodies, antigens and enzymes, retain biological activity very well in the dried state. In the dried condition, the reagents are usually stable for storage even in room temperature. Thus, there is no need to maintain a strict cold chain in reagent supply logistics. This reduces costs of shipping and storage. Dried reagents also allow the design of simpler test instruments for point-of-care use.
It is also of common knowledge that the dried bioaffinity reagents must be kept hermetically closed to avoid contact with ambient moisture. Upon exposure to moisture, the dried reagents tend to loose biological activity, which leads to decrease in assay performance. In case the assay reagents are dried in the final reaction cuvette, the reaction cuvette must be sealed hermetically to avoid contact with ambient humidity. Most often this is realized with an adhesive metal foil. To improve the mechanical properties, the foil can be composed of several co-layers of variable materials. A common type of foil is composed of a plastic layer and a metal foil layer. The plastics layer makes the foil more durable and flexible. In case hermetic sealing is not needed, the reaction cuvette can be sealed with a bare plastic film to protect from dust and other occasional spillovers.
In a typical automated IVD analyser using dried reagents, the clinical sample can be dispensed through the cover foil to the reaction cuvette by a dispensing needle. The dispensed sample dissolves the dried reagents, and triggers the binding reaction between the analyte and the reagents. Mixing or shaking of the reaction cuvette is often needed to accelerate dissolution of the reagents and to enhance reaction kinetics. In point-of-care settings, fast reaction kinetics is essential due to the requirement for a short turn-around-time. In most analysers, subsequent processing of the reaction well is usually needed, such as washing of the unbound components and addition of components that allow quantitation of immunoassay binding degree (e.g. substrate or enhancement solution). Thus, the well needs to be accessed several times.
Shaking of open reaction cuvettes tends to cause spill over and aerosol formation, which can lead to contamination of proximate reaction cuvettes. This can cause false test results, and deteriorate both accuracy and imprecision of the test method. Mechanical mixing is thus associated with a significant carry over risk.
In case of miniaturized test systems where the reaction volume is small, evaporation of the solvent from an open cuvette may also play a role to a significant degree. In such a case the actual concentrations increase, which distorts the assay results. In miniaturized systems, the effects of spill over and aerosol formation are pronounced in comparison to conventionally sized cuvettes.
Evaporation and spilling caused by shaking could be avoided by sealing of test cuvettes after dispensing of the sample. Sealing of the cuvettes, however, would complicate the manual test protocol or, if the method was automated, it would significantly complicate the design of the analyser. In conclusion, a sealing step should be avoided to make the analyser suited for routine IVD use at the point-of-care.
If the cuvette was covered with a foil (or other type of cover) and the dispensing of the samples is carried out through the foil with a thin dispensing needle, probability for spilling would be decreased when compared to open cuvettes. In such a case, the probability of spilling would be proportional to the diameter of the piercing needle. However, even in this case, spilling is very likely to occur during shaking and significant evaporation is likely to occur during incubation. These can deteriorate assay performance.
Re-Sealable Piercable Covers
In order to overcome the problems described above, the cuvettes could be sealed with a re-sealing piercable cover. Many kind of re-sealing covers are known in the art. These covers can be made of plastic films or of flexible materials, such as rubber, silicon, and other elastomers. Such covers are widely applied to cover, for example, reaction vials of nucleic acid amplification reactions, such as thermocycled PCR reactions. In these, the sealing is typically pierced after the cycling to aspirate the liquid. These covers, however, are hardy applicable to miniature reaction cuvettes, such as microtitration wells of the 384 well format. One of the major obstacles with such elastomer covers is the increase of air pressure in the cuvette due to the dispensing. In order to avoid the increased pressure, an equivalent volume of air should flow out of the cuvette. In case of a rubber or a silicon cover, the dispensing needle sits tightly in the pierced opening, and does not let air flow out. The increased pressure impairs the accuracy of dispensing, or it can fail the dispensing completely. In conclusion, piercable covers made of moulded rubber, silicon, or other resilient/elastic bulk material, are not well suited to cover small volume reaction cuvettes.
The problems of increased pressure can be overcome by pre-scoring (pre-slitting) the sealing material at the expected piercing point. Pre-scoring can be of linear shape, Y-shape, or cross-shape or other. Upon piercing with a needle, the edges of the score would bend downwards, thus opening a cleavage for free air outflow. After retraction of the needle, the edges must revert to their original position to close the opening properly. Therefore, the cover material must be elastic and/or resilient. Complete pre-scoring of the cover material allows free diffusion of ambient gases to the cuvette, thus closing is not hermetic. Accordingly, completely pre-scored sealers are not applicable as such with dried reagents.
The elastic cover, whether pre-scored or not, can be topped with a metal layer to keep the cover hermetic until pierced with a needle. Such cover materials are commonly used to pouch microtitration plates, strips and other moisture sensitive bioassay consumables. The metal layer, however, is inelastic. Thus it resists the bending of the slit edges. Once the edges are bent down due to piercing, the metal layer resists recovery of the edges to their original position. In other words, the metal foil disturbs proper reversible function of the pre-scored elastomer cover. If the opening does not close properly, it can lead to spilling or evaporation of the reaction mixture. This again deteriorates method performance.
None of the prior art methods for sealing of reaction cuvettes fulfil criteria for being:    (i) hermetic during storage    (ii) allowing accurate dispensing with a piercing needle    (iii) allowing outflow of air during dispensing    (iv) reversibly closing the pierced opening to avoid spilling and evaporation